Effects of changes of intracellular pH on contraction in sheep cardiac Purkinje fibers

Intracellular pH (pHi) was measured with a pH-sensitive microelectrode in voltage-clamped sheep cardiac Purkinje fibers while tension was simultaneously measured. All solutions were nominally CO2/HCO3 free and were buffered with Tris. The addition of NH4Cl (5-20 mM) produced an initial intracellular alkalosis that was associated with an increase of twitch tension. At the same time, a component of voltage-dependent tonic tension developed. Prolonged exposure (greater than 5 min) to NH4Cl resulted in a slow recovery of pHi accompanied by a decrease of tension. Removal of NH4Cl produced a transient acidosis that was accompanied by a fall of force. In some experiments, there was then a transient recovery of force. If extracellular pH (pHo) was decreased, then pHi decreased slowly. Tension also fell slowly. An increase of pHo produced a corresponding increase of both force and pHi. The application of strophanthidin (10 microM) increased force and produced an intracellular acidosis. The addition of NH4Cl, to remove this acidosis partially, produced a significant increase of force. The above results show that contraction is sensitive to changes of intracellular but not extracellular pH. This pH dependence will therefore modify the contractile response to inotropic maneuvers that also affect pHi.     

Beta 1 and beta 2 adrenergic agonists in exocrine pancreatic secretion in the rabbit

The effect of Dobutamine (a beta 1-adrenergic agonist) and Terbutaline (a beta 2-adrenergic agonist) on exocrine pancreatic secretion was studied in anaesthetized rabbits, simultaneously controlling pancreatic blood flow and blood pressure. The secretion of fluid and ions (bicarbonate, sodium and potassium) was unaffected by the infusion of Dobutamine (8 micrograms.kg-1.min-1) or Terbutaline (10 micrograms.kg-1.min-1). Neither were pancreatic blood flow or mean blood pressure altered. Dobutamine or Terbutaline depress the function of the acinar cells, amylase secretion being more affected by the action of Terbutaline. The results show that beta 1 and beta 2-adrenergic stimulation has no effect on the ductular cells but does decrease the secretion by the acinar cells.     

Characterization of the helicase activity of the Escherichia coli RecBCD enzyme using a novel helicase assay

We describe an assay to measure the extent of enzymatic unwinding of DNA by a DNA helicase. This assay takes advantage of the quenching of the intrinsic protein fluorescence of Escherichia coli SSB protein upon binding to ssDNA and is used to characterize the DNA unwinding activity of recBCD enzyme. Unwinding in this assay is dependent on the presence of recBCD enzyme and linear dsDNA, is consistent with the known properties of recBCD enzyme, and closely parallels other methods for measuring recBCD enzyme helicase activity. The effects of varying temperature, substrate concentrations, enzyme concentration, and mono- and divalent salt concentrations on the helicase activity of recBCD enzyme were characterized. The apparent Km values for recBCD enzyme helicase activity on linear M13 dsDNA molecules at 25 degrees C are 0.6 nM dsDNA molecules and 130 microM ATP, respectively. The apparent turnover number for unwinding is approximately 15 microM base pairs s-1 (microM recBCD enzyme)-1. When this rate is corrected for the observed stoichiometry of recBCD enzyme binding to dsDNA, kcat for helicase activity corresponds to an unwinding rate of approximately 250 base pairs of DNA s-1 (functional recBCD complex)-1 at 25 degrees C. At 37 degrees C, the apparent Km value for dsDNA molecules was the same as that at 25 degrees C, but the apparent turnover number became 56 microM base pairs s-1 (microM recBCD enzyme)-1 [or 930 base pairs s-1 (functional recBCD complex)-1 when corrected for observed stoichiometry]. With increasing NaCl concentration, kcat peaks at 100 mM, and the apparent Km value for dsDNA increases by 3-fold at 200 mM NaCl. In the presence of 5 mM calcium acetate, the apparent Km value is increased by 3-fold, and kcat decreased by 20-30%. We have also shown that recBCD enzyme molecules are able to catalytically unwind additional dsDNA substrates subsequent to initiation, unwinding, and dissociation from a previous dsDNA molecule.     

Alterations in the regulatory region of the human papillomavirus type 6 genome are generated during propagation in Escherichia coli.

We analyzed the long control regions (LCRs) of seven human papillomavirus type 6b (HPV-6b) clones, which contained prototype HPV-6b sequences recloned into various plasmid vectors and propagated in different strains of Escherichia coli. Southern blot analysis and DNA sequencing demonstrated three different sequences, each distinct from the published prototype HPV-6b sequence. Two of the plasmids contained insertions of 24 and 94 base pairs (bp) and a 1-bp deletion. Four plasmids contained insertions of 24 and 58 bp and a deletion of 49 bp. One plasmid contained a single insertion of 77 bp. The 94-, and 58-bp insertions occurred at the same site and had 100% positional identity across their shared lengths. All changes were located in the purine-thymidine-rich region of the LCR (nucleotides 7292 to 7400). Two additional LCR sequences were detected by restriction analysis of two other HPV-6b clones. We conclude that the purine-thymidine-rich region of the LCR is a hot spot for recombination in E. coli and that the alterations are the result of recA-independent events. These results emphasize the need to rigorously prove that a cloned isolate is an authentic copy of the genomic DNA present in the original lesion. In addition, the data indicate that the HPV-6b LCR sequences employed in different laboratories may be different, even if their parental DNAs were identical. Finally, we discuss the need for caution in assigning biological significance to alterations in this region, in view of the limited data available on the true identity of the HPV-6b LCR.     

Evidence for a non-opioid sigma binding site in the guinea-pig myenteric plexus

The presence of a binding site to (+)-(3H)SKF 10,047 was demonstrated in a guinea-pig myenteric plexus (MYP) membrane preparation. Specific binding to this receptor was saturable, reversible, linear with protein concentration and consisted of two components, a high affinity site (KD = 46 +/- 5 nM; Bmax = 3.4 +/- 0.5 pmole/g wet weight) and a low affinity site (KD= = 342 +/- 72 nM; Bmax = 22 +/- 3 pmole/g wet weight). Morphine and naloxone 10(-4) M were unable to displace (+)-(3H)SKF 10,047 binding. Haloperidol, imipramine, ethylketocyclazocine and propranolol were among the most potent compounds to inhibit this specific binding. These results suggest the presence of a non-opioid haloperidol sensitive sigma receptor in the MYP of the guinea-pig.